Amino acids currently have application as additives to animal feed, nutritional supplements for human food, components in infusion solutions, and synthetic intermediates for the manufacture of pharmaceuticals and agricultural chemicals. L-glutamic acid is used as a flavor enhancer for food with a world market of over 1 billion dollars annually. L-lysine and methionine are large volume additives to animal feed, and L-tryptophan and L-threonine have similar potential applications. L-phenylalanine and L-aspartic acid have very important markets as key components in the manufacture of the sweetener aspartame. Infusion solutions require a range of amino acids including those essential in human diets.
Methods currently in use for the production of amino acids include extraction, chemical synthesis followed by resolution, fermentation and enzymatic synthesis (biocatalysis). Extraction procedures require extensive purification of the amino acid of interest from protein hydrolyzates. With chemical synthetic methods, normally a racemic mixture is formed, and the resolution to produce the optically active product is often costly and inefficient. Fermentation, while overcoming many of the disadvantages inherent in the previously mentioned methods, suffers from problems of slow rates of conversion, dilute solutions, costly purifications, and very high capital costs. Biocatalysis offers the potential for lower cost production in many cases primarily because of the significantly reduced capital requirements, lower purification costs due to the absence byproducts in the product stream, and higher rates of conversion of substrates to products because fewer enzymatic steps are involved.
Some biocatalytic processes are currently in use. For example, L-aspartic acid is produced in commercial quantities by the reaction of fumaric acid with ammonia in the presence of the enzyme aspartase. See Tosa et al, Appl. Microbiol. 27, 886-9 (1974). L-phenylalanine can be produced by enzymatic synthesis from cinnamic acid and ammonia using the enzyme phenylalanine-ammonia lyase. L-alanine can be synthesized from L-aspartic acid by enzymatic decarboxylation. See. U.S. Pat. Nos. 3,458,400 and 3,463,704. These processes are useful for the production of the individual amino acids listed. However, none of these processes is based on a general enzymatic technology broadly applicable to the production of many amino acids.
One enzymatic process broadly applicable to the production of many amino acids is described in U.S. Pat. No. 3,183,170. In the process of U.S. Pat. No. 3,183,170 L-glutamic acid and a keto acid are combined with transaminase to produce alpha-keto glutaric acid and L-amino acid. The alpha-keto glutaric acid is continuously reduced to L-glutamic acid in the presence of a multi enzyme system, hydrogen gas, and a nitrogen souce, such as inorganic ammonium salt, organic ammonium salt, ammonium hydroxide, ammonia gas, or urea. The L-glutamic acid thus produced is recycled thus enabling the production of large amounts of L-amino acid with a small quantity of L-glutamic acid. However, this multienzyme system is difficult to operate on a commercial scale because it requires the cofactor NAD/NADH which is expensive, hydrolytically unstable and sensitive to oxygen and to light. Thus, a general enzymatic technique broadly applicable to the production of many amino acids is still desired.